Hepatitis B virus (HBV) is the etiological agent of a wide spectrum of clinical abnormalities and is a major public health problem worldwide. The spectrum of disease associated with HBV includes acute self-limiting infections, life-threatening fulminant hepatitis, and chronic hepatitis which can progress to cirrhosis and lead to liver failure. Furthermore, chronic infection has been epidemiologically associated with development of hepatocellular carcinoma. In areas such as Southeast Asia, China and sub-Saharan Africa where HBV infection is endemic, the proportion of chronically infected individuals may range from 5-20% of the adult population. The number of chronically infected HBV carriers worldwide has been estimated to be 200 million. The risk to these individuals of developing hepatocellular carcinoma has been estimated to be as high as 15%. Long-term consequences of HBV infection can lead to mortality in 60% of infected individuals in some populations.
Therapy for acute or chronic HBV infections is currently inadequate and has mainly been limited to supportive therapy. Experimental therapies using antiviral drugs such as adenosine arabinoside (araA) and interferon-a have proven effective in suppressing HBV replication in chronically infected individuals. However, permanent suppression after discontinuation of antiviral therapy occurs in only a small percentage of treated patients. Significant clinical benefits require an anti-HBV drug which could be administered continuously without side effects or which could eliminate HBV replication permanently following a course of therapy. An obvious need exists for a clinically effective antiviral therapy for acute and chronic HBV infections. Such an antiviral would also be useful for treating individuals accidently exposed to clinical specimens or blood products containing infectious HBV, to prevent the development of HBV-associated disease. There is also a need for research reagents and diagnostics which are able to differentiate HBV from other agents causing hepatitis and which are useful in designing appropriate therapeutic regimes.
Antisense oligonucleotides PA0 HBV replication PA0 Identification of HBV targets for antisense therapy PA0 Antisense oligonucleotides PA0 Pharmacokinetics of antisense oligonucleotides PA0 In vitro evaluation of HBV antisense oligonucleotides PA0 Animal models for HBV
Oligonucleotides are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which, by nature, are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes, for example to determine which viral genes are essential for replication, or to distinguish between the functions of various members of a biological pathway. This specific inhibitory effect has, therefore, been harnessed for research use. This specificity and sensitivity is also harnessed by those of skill in the art for diagnostic uses. Viruses capable of causing similar hepatic symptoms can be easily and readily distinguished in patient samples, allowing proper treatment to be implemented. Antisense oligonucleotide inhibition of viral replication in vitro is useful as a means to determine a proper course of therapeutic treatment. For example, before a patient suspected of having an HBV infection is treated with an oligonucleotide composition of the present invention, cells, tissues or a bodily fluid from the patient can be contacted with the oligonucleotide and inhibition of viral replication can be assayed. Effective in vitro inhibition of HBV replication indicates that the infection will be responsive to the oligonucleotide treatment.
Oligonucleotides have been employed as drugs for the treatment of disease states in animals and man. For example, workers in the field have now identified antisense, triplex and other oligonucleotide compositions which are capable of modulating expression of genes implicated in viral, fungal and metabolic diseases.
As examples, U.S. Pat. No. 5,166,195 issued Nov. 24, 1992 provides oligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810, issued Apr. 2, 1991, provides oligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA and inhibiting replication. U.S. Pat. No. 5,194,428, issued Mar. 16, 1993, provides antisense oligonucleotides having antiviral activity against influenzavirus. U.S. Pat. No. 4,806,463, issued Feb. 21, 1989, provides antisense oligonucleotides and methods using them to inhibit HTLV-III replication. U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423 (Cohen et al.) are directed to phosphorothioate oligonucleotide analogs used to prevent replication of foreign nucleic acids in cells. Antisense oligonucleotides have been safely administered to humans and clinical trials of several antisense oligonucleotide drugs are presently underway. The phosphorothioate oligonucleotide, ISIS 2922, has been shown to be effective against cytomegalovirus retinitis in AIDS patients, BioWorld Today, Apr. 29, 1994, p. 3. It is thus established that oligonucleotides can be useful drugs for treatment of cells and animal subjects, especially humans.
Blum et al. (Lancet 1991, 337, 1230) showed inhibition of hepatitis B virus antigens in transfected hepatocytes by an undisclosed antisense oligodeoxynucleotide which blocked HBsAg and HBeAg synthesis as well as HBV replication.
Blum et al. (PCT publication WO 94/24864) subsequently disclosed termination of hepatitis B replication by antisense oligonucleotides complementary to mRNA which is complementary to a portion of the minus strand of the viral genome which encodes the terminal protein region of the viral polymerase.
Goodarzi et al. (J. Gen. Virol 1990, 71, 3021-3025) tested the effects of a number of 12- to 15-mer phosphodiester oligonucleotides on the expression of the HBV HBsAg surface antigen in an HBV-infected human cell line. They found that oligonucleotides directed against the cap site and region around the initiation site were most effective, giving inhibition of up to 96% at an oligonucleotide concentration of 17.4 .mu.M. A phosphorothioate analog of one of the most active sequences was also tested and gave 90% inhibition of HBsAg expression at a concentration of 5.8 .mu.M.
Wu and Wu (J. Biol. Chem. 1992, 267,12436-12439) used a 21-mer oligodeoxynucleotide complementary to the polyadenylation signal for HBV, coupled to an asialoglycoprotein targeting moiety. In the infected cell line HepG2, treatment with oligonucleotide-asialoglycoprotein complex at an oligonucleotide concentration of 50 .mu.M resulted in 80% inhibition of HBsAg expression after one day and an 80% decrease in HBV DNA. In the presence of uncomplexed antisense oligonucleotide at a concentration of 50 .mu.M, HBsAg concentrations continued to rise steadily throughout the 7 days of treatment, though after three days of treatment the treated cells had 30% less antigen than controls.
Yao et al. examined the effect of antisense phosphorothioate oligodeoxynucleotides on HBsAg and HBeAg production (Yao et al., Chung Hua I Hsueh Tsa Chih 1994, 74, 74-76).
Offensperger et al. (EMBO J. 1993, 12, 1257-1262) used phosphorothioate antisense oligonucleotides to inhibit duck hepatitis B virus (DHBV) in cultured duck hepatocytes. Nine oligonucleotides were tested, of which four were targeted to the pre-S/S region, one to the start of the polymerase region and four to the pre-C/C region. All showed some activity and two, directed to the start of the pre-S region and the direct repeat (DR)II region, were particularly active. The active oligonucleotide directed to the start of the pre-S region was tested on DHBV-infected ducklings in vivo. Oligonucleotide was injected intravenously daily for ten days, after which the livers were analyzed for DHBV. All ducklings showed a nearly complete inhibition of viral replication after oligonucleotide treatment, and no hepatotoxicity was detected. Two DHBV-negative ducklings were also treated with the same oligonucleotide and subsequently infected by injection of DHBV. These ducks were found not to be infected with DHBV twelve days later, showing that the oligonucleotide treatment was able to prevent infection.